Antiviral nucleoside combination

ABSTRACT

Synergistic combinations of nucleoside derivatives, pharmaceutical formulations containing said combinations and use of the combinations in the treatment of retroviral infections are disclosed.

This is a divisional of application Ser. No. 07/846,367 filed on Mar. 5,1992 now U.S. Pat. No. 5,234,913.

The present invention relates to synergistic antiviral combinations ofnucleoside derivatives, pharmaceutical formulations containing saidcombinations and their use in medical therapy, particularly in thetreatment of virus infections, especially retrovirus infections.

Acquired immunodeficiency syndrome, (AIDS) is an immunosuppressive orimmunodestructive disease that predisposes subjects to fatalopportunistic infections. Characteristically, AIDS is associated with aprogressive depletion of T-cells, especially the helper-inducer subsetbearing the OKT⁴ surface marker.

Human immunodeficiency virus (HIV) has been reproducibly isolated frompatients with AIDS or with the symptoms that frequently precede AIDS.HIV is cytopathic and appears to preferentially infect and destroyT-cells bearing the OKT⁴ marker. It is now generally recognized that HIVis the etiological agent of AIDS.

Since the discovery that HIV is the etiological agent of AIDS, numerousproposals have been made for anti-HIV chemotherapeutic agents that maybe effective in treating AIDS sufferers. Thus, for example, EuropeanPatent Specification 0 382 526 describes anti-HIV substituted1,3-oxathiolanes. U.S. Patent Specification 4724232 and EuropeanSpecification 0 196 185 describe 31-azido-3'-deoxythymidine (which hasthe approved name zidovudine) and its use in treating AIDS.

Since the priority date of this patent application, the following itemshave been published: Liotta, D. C. and Choi, W. B., Synthesis of BCH-189and related compounds, PCT Appl. WO 91/11186; Soudeyns, H. et al.,Anti-human immunodeficiency virus type 1 activity and in vitro toxicityof 2'-deoxy-3'-thiacytidine (BCH-189), Antimicrob. Agents Chemother., 35(7), 1386-90 (1991); Choi, W. B. et al., In situ complexation directsthe stereochemistry of N-glycosylation in the synthesis of thialanyl anddioxolanyl nucleoside analogs, J. Am. Chem. Soc., 113(24), 9377-9(1991).

We have now discovered that1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine incombination with 31-azido-3'-deoxythymidine (zidovudine) results in asurprisingly large potentiation of the anti-HIV activity of thecompounds. The use of1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine inconjunction with zidovudine produces a synergistic increase in anti-HIVactivity in comparison with the anti-HIV activities of the individualcompounds.

According to a first feature of the present invention there is provideda combination of (a)1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine of formula(I): ##STR1## or a physiologically functional derivative thereof and (b)3'-azido-3'-deoxythymidine (zidovudine) of formula (II): ##STR2## or aphysiologically functional derivative thereof, components (a) and (b) ofthe combination being employed together such that a synergisticantiviral effect is achieved. The term "synergistic antiviral effect" isused herein to denote an antiviral effect which is greater than thepredicted purely additive effects of the individual components (a) and(b) of the combination.

It should be noted that the compound of formula (I) contains two chiralcenters and therefore exists in the form of two pairs of optical isomers(i.e. enantiomers) and mixtures thereof including racemic mixtures.Thus, the compound of formula (I) may be either a cis or a trans isomeror mixtures thereof. Each cis or a trans isomer or mixtures thereof.Each cis and trans isomer can exist as one of two enantiomers ormixtures thereof including racemic mixtures.

All such isomers and mixtures thereof including racemic mixtures arewithin the scope of the invention which also includes the tautomericforms of the compounds of formula (I) and (II). The cis isomers of thecompound of formula (I) are preferred.

By "physiologically functional derivative" is meant a pharmaceuticallyacceptable salt, ester or salt of an ester of the parent compound offormula (I) or (II), a pharmaceutically acceptable amide of the compoundof (I), or any other compound which upon administration to the recipientis capable of providing (directly or indirectly) the parent compound oran active metabolite or residue thereof.

Preferred esters according to the invention include carboxylic acidesters in which the non-carbonyl moiety of the ester grouping isselected from straight or branched chain alkyl e.g. n-propyl, t-butyl,n-butyl, alkoxyalkyl (e.g. methoxymethyl), aralkyl (e.g. benzyl),aryloxyalkyl (e.g. phenoxymethyl), and aryl (e.g. phenyl); sulfonateesters such as alkyl- or aralkylsulfonyl (e.g. methanesulfonyl); aminoacid esters (e.g. L-valyl or L- isoleucyl); dicarboxylic acid esters(e.g., hemisuccinate); and mono-, di- or triphosphate esters. Thephosphate esters may be further esterified by, for example, a C₁₋₂₀alcohol or reactive derivative thereof, or by a 2,3-di(C₆₋₂₄)acylglycerol.

Any alkyl moiety present in such esters advantageously contains 1 to 18carbon atoms, particularly 1l to 4 carbon atoms. Any aryl moiety presentin such esters advantageously comprises a phenyl group optionallysubstituted, e.g. by halogen, C₁₋₄ alkyl, C₁₋₄ alkoxy or nitro.

The above-mentioned pharmaceutically acceptable amides of the compoundof formula (I) include those derivatives wherein the cytosine aminogroup is present in the form of an amide, eg. NHCOR wherein R is C₁₋₆alkyl or aryl (eg. phenyl optionally substituted by halogen, C₁₋₄ alkyl,C₁₋₄ alkoxy, nitro or hydroxyl).

Examples of pharmaceutically acceptable salts include base salts, e.g.derived from an appropriate base, such as alkali metal (e.g. sodium),alkaline earth metal (e.g. magnesium) salts, ammonium and NX₄ ⁺ (whereinX is C₁₋₄ alkyl). Pharmaceutically acceptable acid addition saltsinclude salts of organic carboxylic acids such as acetic, lactic,tartaric, malic, isethionic, lactobionic and succinic acids; organicsulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonicand p-toluenesulfonic acids and inorganic acids such as hydrochloric,sulfuric, phosphoric and sulfamic acids.

Examples of viral infections and associated clinical conditions whichmay be treated or prevented in accordance with the invention, includehuman retroviral infections such as human immunodeficiency virus (HIV),e.g. HIV-1 or HIV-2, and human T-cell lymphotropic virus (HTLV), e.g.HTLV-I or HTLV-II infections. The combinations of the present inventionare especially useful for the treatment of AIDS and related clinicalconditions such as AIDS-related complex (ARC), progressive generalizedlymphadenopathy (PGL), AIDS-related neurological conditions, such asmultiple sclerosis or tropical paraparesis, anti-HIV antibody-positiveand HIV-positive conditions such as thrombocytopenic purpura. Thecombinations of the present invention may also be used in the treatmentof psoriasis. The combinations of the present invention have been foundto be particularly applicable to the treatment of asymptomaticinfections or diseases caused by or associated with human retroviruses.

According to a second feature of the invention there are providedcombinations as hereinbefore described, for use in medical therapy,particularly for the treatment or prophylaxis of any of theaforementioned viral infections or conditions, especially HIV infectionsincluding AIDS.

The present invention further includes a process for preparing thecombinations hereinbefore described, which comprises bringing intoassociation components (a) and (b) of the combination in a medicament toprovide a synergistic antiviral effect.

In a further aspect of the present invention, there is provided the useof a combination of the present invention in the manufacture of amedicament for the treatment of any of the aforementioned viralinfections or conditions.

The present invention further provides a method for the treatment orprophylaxis of viral infections (especially HIV infections) in a mammal(including a human) which comprises administering to said mammal aneffective amount of a combination as hereinbefore described. It will beappreciated that in accordance with the present invention, components(a) and (b) of the combination may be administered simultaneously orsequentially. In the latter case, however, the components areadministered within a sufficiently short interval to ensure that asynergistic antiviral effect is achieved.

The present invention also provides a method of potentiating in a mammal(including a human) having a viral infection, the antiviral activitiesof components (a) and (b) of the combination, which comprisesadministering to said mammal an effective synergistic amount ofcomponent (a) simultaneously with, previous to, or subsequent to theadministration of component (b).

An advantage of the combination of the present invention is that itenables attainment of an improved antiviral efficacy at a particulardose of one of the antiviral components (compared with the componentused alone) thereby improving the therapeutic index of the component.Thus, for example, the combination may be used to treat conditions whichwould otherwise require relatively large doses of the antiviralcomponent at which toxicity problems may occur. The smaller doses of thecombination may provide increased convenience to the recipient andincreased compliance.

The combinations of the present invention may be administered to amammal in a conventional manner. As indicated above, components (a) and(b) may be administered simultaneously (e.g., in a unitarypharmaceutical formulation) or separately (e.g., in separatepharmaceutical formulations). In general, the combinations may beadministered by the topical, oral, rectal or parenteral (e.g.,intravenous, subcutaneous or intramuscular) routes. It will beappreciated that the route may vary with, for example, the severity ofthe condition to the treated and the identity of the recipient.

It will be appreciated that, while there will usually be an optimumratio of the components to ensure maximum potentiation, even avanishingly small quantity of one component will suffice to potentiatethe effect of the other to some degree, and so any ratio of twopotentiating components will still possess the required synergisticeffect. However, greatest synergy is generally observed when the twocomponents are present in particular ratios.

Thus the optimum molar ratios of zidovudine to1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine, or theirrespective physiologically functional derivatives, for use according tothis invention are from 1:1 to 1:600, preferably from 1:10 to 1:250, andmost preferably 1:25.

Hereafter the components of the combination may be referred to as"active ingredients".

The dose of the combination will depend on the condition being treatedand other clinical factors such as the weight and condition of therecipient and the route of administration of the components of thecombinations. Examples of dose ranges and component ratios are asfollows:

In general a suitable dose of a combination of the present inventionbased on the total weight of components (a) and (b) will be in the rangeof 3 to 120 mg per kilogram body weight of the recipient per day,preferably in the range of 6 to 90 mg per kilogram body weight per dayand most preferably in the range 15 to 60 mg per kilogram body weightper day. The desired dose is preferably presented as two, three, four,five, six or more sub-doses administered at appropriate intervalsthroughout the day. These sub-doses may be administered in unit doseforms, for example, containing 10 to 1500 mg, preferably 20 to 1000 mg,and most preferably 50 to 700 mg of active ingredients per unit doseform.

While it is possible for the active ingredients to be administered aloneit is preferable to present them as pharmaceutical formulations.Pharmaceutical formulations of the present invention comprise acombination according to the invention together with one or morepharmaceutically acceptable carriers or excipients and optionally othertherapeutic agents. When the individual components of the combinationare administered separately they are generally each presented as apharmaceutical formulation. The references hereinafter to formulationsrefer unless otherwise stated to formulations containing either thecombination or a component thereof. Formulations include those suitablefor oral, rectal, nasal, topical (including transdermal, buccal andsublingual), vaginal or parenteral (including subcutaneous,intramuscular, intravenous and intradermal) administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. Suchmethods include the step of bringing into association the activeingredients with the carrier which constitutes one or more accessoryingredients.

In general, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then if necessary shaping theproduct.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredients; as apowder or granules; as a solution or a suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredients in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g. sodium starchglycollate, cross-linked povidone, cross-linked sodium carboxymethylcellulose) surface-active or dispersing agent. Molded tablets may bemade by molding a mixture of the powdered compound moistened with aninert liquid diluent in a suitable machine. The tablets may optionallybe coated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredients therein using, for example,hydroxypropylmethyl cellulose in varying proportions to provide thedesired release profile. Tablets may optionally be provided with anenteric coating, to provide release in parts of the gut other than thestomach.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredients in a flavored base, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier. Formulations for rectal administration may bepresented as a suppository with a suitable base comprising, for example,cocoa butter or a salicylate.

Topical administration may also be by means of a transdermaliontophoretic device.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnonaqueous isotonic sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents; and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. The formulations may be presented in unit-dose ormulti-dose sealed containers, for example, ampules and vials, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules andtablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose ordaily subdose of the active ingredients, as hereinbefore recited, or anappropriate fraction thereof.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude such further agents as sweeteners, thickeners and flavoringagents.

The compounds of the combination of the present invention may beprepared in conventional manner. Zidovudine can be prepared, forexample, as described in U.S. Pat. No. 4,724,232, incorporated herein byreference. Zidovudine can also be obtained from Aldrich Chemical Co.,Milwaukee, Wis. 53233, U.S.A.

1-(2-(Hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine may beprepared for example by:

a) reacting optionally protected 5-fluorocytosine with a 1,3-oxathiolaneof formula (IIIA): ##STR3## wherein R₁ is hydrogen or a hydroxyprotecting group and L is a leaving group; or

b) reacting a compound of formula (IIIB): ##STR4## (wherein R₁ is asdefined above and R₁ ^(a) is an amino protecting group) with afluorinating agent serving to introduce a fluorine atom in the5-position of the cytosine ring; or

c) reacting a compound of formula (IIIC): ##STR5## (wherein R₁ is asdefined above) with an agent serving to convert the oxo group in the4-position of the uracil ring to an amino group; any remainingprotecting groups being removed to produce the desired product.

With the regard to process a), the hydroxy protecting group includesprotecting groups such as acyl (e.g. acetyl), arylacyl (e.g. benzoyl orsubstituted benzoyl), trityl or monomethoxytrityl, benzyl or substitutedbenzyl, trialkylsilyl (e.g. dimethyl-t-butylsilyl) ordiphenylmethylsilyl. The 5-fluorocytosine compound may be optionallyprotected with silyl, e.g. trimethyl silyl groups. Such groups may beremoved in conventional manner. The leaving group L is a leaving grouptypical of those known in the art of nucleoside chemistry e.g. halogensuch as chlorine or bromine, alkoxy such as methoxy or ethoxy or acylsuch as acetyl or benzoyl.

The reaction in process a) may be affected in an organic solvent (e.g.1,2-dichloroethane or acetonitrile) in the presence of a Lewis acid suchas stannic chloride or trimethylsilyl triflate.

Compounds of formula (IIIA) may be obtained from a suitably protected2-hydroxyacetaldehyde of formula (IV):

    R.sub.1 OCH.sub.2 CHO                                      (IV)

wherein R₁ is defined above, as described in Can. J. Research, 8, 129(1933) and European Patent Specification 0 382 526. Reaction ofcompounds of formula IV with a mercaptoacetal HSCH₂ CH(OR)₂ wherein R isC₁₋₄ alkoxy such as HSCH₂ CH(OC₂ H₅ ₂, known in the art (Chem. Ber. 85,924-932 (1952)), yields compounds of formula (IIIA) wherein L is OR(alkoxy) e.g. methoxy or ethoxy. Alternatively, compounds of formulaIIIA, wherein L is alkoxy, may be converted to compounds of formula IIIAwherein L is halogen or acyl by methods known in the art of carbohydratechemistry.

Compounds of formula (IV) may be prepared from 1,2-O-isopropylideneglycerol by introduction of R₁ (e.g. trisubstituted silyl, benzyl ortrityl) and removal of the isopropylidene group with mild acid (e.g.aqueous formic or acetic acid) or zinc bromide in acetonitrile, followedby oxidation of the alcohol group with aqueous periodate.

With regard to process b), the 5-fluoro substituent may be introduced bymethods known in the art (M. J. Robins, et al., in Nucleic AcidChemistry. Part 2, L. B. Townsend and R. S. Tipson, editors, J. Wileyand Sons, New York, 895-900 (1978) and references therein; R. Duschinskyin Nucleic Acid Chemistry, Part 1, L. B. Townsend and R. S. Tipson,editors, J. Wiley and Sons, New York, 43-46 (1978) and referencestherein). The fluorinating agent may be, for example,trimethylhypofluorite in fluorotrichloromethane.

With regard to process c), the compound of formula (IIIC) isadvantageously treated with 1,2,4-triazole, advantageously together with4-chlorophenyl dichlorophosphate, to form the corresponding4-(1,2,4-triazolyl) compound which is then converted to the desired4-amino (cytidine) compound by reaction with for example methanol.

The starting materials of formulas (IIIB) and (IIIC) may be prepared forexample by reaction of an appropriate (optionally protected) base with acompound of formula (IIIA) in an analogous manner to that described inprocess a). 5-Fluorouracil and 5-fluorocytosine are commerciallyavailable from Aldrich Chemical Co., Milwaukee, Wis. 53233, U.S.A.

Separation of the (±)-cis and (±)-trans isomers of formula (I) forexample in a protected form, may be accomplished by chromatography onsilica gel with mixtures of organic solvents such as ethylacetate/methanol, ethyl acetate/hexane or dichloromethane/methanol. Anyprotecting group may then be removed using the appropriate reagent foreach group.

Esters of the component compounds of formulas (I) and (II) may beprepared in conventional manner by reaction with an appropriateesterifying agent such as an acid halide or anhydride. The compounds offormulas I and II or esters thereof may be converted intopharmaceutically acceptable salts thereof by treatment with anappropriate base. An ester or salt of the component compounds may beconverted into the parent compound by hydrolysis.

Pharmaceutically acceptable amides of the compound of formula (I) may beprepared, for example by reaction with an appropriate acylating agent,for example, an acid halide or anhydride serving to acylate the 5'-OHand 4-NH₂ groups. The acyl group may then be removed selectively fromone or other of the 5'-OH and 4-NH₂ groups. For example, treatment ofthe diacylated compound under acidic conditions, eg. a Lewis acid suchas zinc bromide in methanol, removes the 4N-acyl group to yield thecorresponding 5'-OH ester, while treatment of the diacylated compoundunder alkaline conditions, eg. with sodium methoxide removes the 5'-OHacyl group to yield the corresponding 4N-amide. The acyl groups can alsobe removed selectively by treatment with commercially available esteraseor lipase enzymes, eg. pig liver esterase or pancreatic lipase, or bytreatment in accordance with methods described in U.S. PatentSpecification No. 5071983. The compound of formula (I) may be convertedinto a pharmaceutically acceptable salt thereof in a conventionalmanner, for example, by treatment with an appropriate base.

The following examples are intended for illustration only and are notintended to limit the scope of the invention in any way. "Activeingredient" denotes a mixture of the components zidovudine andcis-1-(2(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine in themolar ratio of 1:25.

EXAMPLE 1

Tablet Formulation

The following formulations A, B and C are prepared by wet granulation ofthe ingredients with a solution of povidone, followed by addition ofmagnesium stearate and compression.

    ______________________________________                                                        mg/tablet                                                     ______________________________________                                        Formulation A                                                                 Active Ingredient 250                                                         Lactose B.P.      210                                                         Povidone B.P.     15                                                          Sodium Starch Glycollate                                                                        20                                                          Magnesium Stearate                                                                              5                                                                             500                                                         Formulation B                                                                 Active Ingredient 250                                                         Lactose B.P.      150                                                         Avicel PH 101     60                                                          Povidone B.P.     15                                                          Sodium Starch Glycollate                                                                        20                                                          Magnesium Stearate                                                                              5                                                                             500                                                         Formulation C                                                                 Active Ingredient 250                                                         Lactose B.P.      200                                                         Starch            50                                                          Povidone          5                                                           Magnesium Stearate                                                                              4                                                                             359                                                         ______________________________________                                    

The following formulations, D and E, are prepared by direct compressionof the admixed ingredients. The lactose in formulation E is of thedirect compression type (Dairy Crest--"Zeparox").

    ______________________________________                                                         mg/tablet                                                    ______________________________________                                        Formulation D                                                                 Active Ingredient  250                                                        Pregelatinized Starch NF15                                                                       150                                                                           400                                                        Formulation E                                                                 Active Ingredient  250                                                        Lactose B.P.       150                                                        Avicel             100                                                                           500                                                        ______________________________________                                    

Formulation F (Controlled Release Formulation)

The formulation is prepared by wet granulation of the ingredients with asolution of povidone followed by the addition of magnesium stearate andcompression.

    ______________________________________                                                          mg/tablet                                                   ______________________________________                                        Active Ingredient   500                                                       Hydroxypropylmethylcellulose                                                                      112                                                       (Methocel K4M Premium)                                                        Lactose B.P.        53                                                        Povidone B.P.       28                                                        Magnesium Stearate  7                                                                             700                                                       ______________________________________                                    

Drug release takes place over a period of about 6-8 hours and iscomplete after 12 hours.

EXAMPLE 2

Capsule Formulations

Formulation A

A capsule formulation is prepared by admixing the ingredients offormulation D in Example 1 above and filling into a two-part hardgelatin capsule. Formulation B (infra) is prepared in a similar manner.

    ______________________________________                                                        mg/capsule                                                    ______________________________________                                        Formulation B                                                                 Active Ingredient 250                                                         Lactose B.P.      143                                                         Sodium Starch Glycollate                                                                        25                                                          Magnesium Stearate                                                                              2                                                                             420                                                         Formulation C                                                                 Active Ingredient 250                                                         Macrogel 4000 B.P.                                                                              350                                                                           600                                                         ______________________________________                                    

Capsules of formulation C are prepared by melting the Macrogel 4000B.P., dispersing the active ingredient in the melt and filling the meltinto a two-part hard gelatin capsule.

    ______________________________________                                                      mg/capsule                                                      ______________________________________                                        Formulation D                                                                 Active Ingredient                                                                             250                                                           Lecithin        100                                                           Arachis Oil     100                                                                           450                                                           ______________________________________                                    

Capsules of formulation D are prepared by dispersing the activeingredient in the lecithin and arachis oil and filling the dispersioninto soft, elastic gelatin capsules.

Formulation E (Controlled Release Capsule)

The following controlled release capsule formulation is prepared byextruding ingredients a, b, and c using an extruder, followed byspheronization of the extrudate and drying. The dried pellets are thencoated with release-controlling membrane (d) and filled into atwo-piece, hard gelatin capsule.

    ______________________________________                                                         mg/capsule                                                   ______________________________________                                        (a) Active Ingredient                                                                            250                                                        (b) Microcrystalline Cellulose                                                                   125                                                        (c) Lactose B.P.   125                                                        (d) Ethyl Cellulose                                                                              13                                                                            513                                                        ______________________________________                                    

EXAMPLE 3

Injectable Formulation

    ______________________________________                                                             mg                                                       ______________________________________                                        Formulation A                                                                 Active Ingredient      200                                                    Hydrochloric Acid Solution 0.1 M or                                                                  4.0 to 7.0                                             Sodium Hydroxide Solution 0.1 M q.s. to pH                                    Sterile water q.s. to  10 ml                                                  ______________________________________                                    

The active ingredient is dissolved in most of the water (351°-40° C.)and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid orthe sodium hydroxide as appropriate. The batch is then made up to volumewith the water and filtered through a sterile micropore filter into asterile 10 ml amber glass vial (type 1) and sealed with sterile closuresand overseals.

    ______________________________________                                        Formulation B                                                                 Active Ingredient        125    mg                                            Sterile, Pyrogen-free, pH 7 Phosphate                                                                  25     ml                                            Buffer, q.s. to                                                               ______________________________________                                    

EXAMPLE 4

Intramuscular injection

    ______________________________________                                        Active Ingredient      200    mg                                              Benzyl Alcohol         0.10   g                                               Glycofurol 75          1.45   g                                               Water for injection q.s. to                                                                          3.00   ml                                              ______________________________________                                    

The active ingredient is dissolved in the glycofurol. The benzyl alcoholis then added and dissolved, and water added to 3 ml. The mixture isthen filtered through a sterile micropore filter and sealed in sterile 3ml amber glass vials (type 1).

EXAMPLE 5

Syrup

    ______________________________________                                        Active Ingredient     250     mg                                              Sorbitol Solution     1.50    g                                               Glycerol              2.00    g                                               Sodium Benzoate       0.005   g                                               Flavor, Peach 17.42.3169                                                                            0.0125  ml                                              Purified Water q.s. to                                                                              5.00    ml                                              ______________________________________                                    

The active ingredient is dissolved in a mixture of the glycerol and mostof the purified water. An aqueous solution of the sodium benzoate isthen added to the solution, followed by addition of the sorbitalsolution and finally the flavor. The volume is made up with purifiedwater and mixed well.

EXAMPLE 6

Suppository

    ______________________________________                                                               mg/capsule                                                                    suppository                                            ______________________________________                                        Active Ingredient        250                                                  Hard Fat, B.P. (Witepsol H15 - Dynamit Nobel)                                                          1770                                                                          2020                                                 ______________________________________                                    

One-fifth of the Witepsol H₁₅ is melted in a steam-jacketed pan at 45°C. maximum. The active ingredient is sifted through a 200 μm sieve andadded to the molten base with mixing, using a Silverson fitted with acutting head, until a smooth dispersion is achieved. Maintaining themixture at 45° C., the remaining Witepsol H15 is added to the suspensionand stirred to ensure a homogenous mix. The entire suspension is passedthrough a 250 μm stainless steel screen and, with continuous stirring,is allowed to cool to 40° C. At a temperature of 38° C. to 40° C., 2.02g of the mixture is filled into suitable, 2 ml plastic molds. Thesuppositories are allowed to cool to room temperature.

EXAMPLE 7

Pessaries

    ______________________________________                                                       mg/pessary                                                     ______________________________________                                        Active Ingredient                                                                              250                                                          Anhydrate Dextrose                                                                             380                                                          Potato Starch    363                                                          Magnesium Stearate                                                                             7                                                                             1000                                                         ______________________________________                                    

The above ingredients are mixed directly and pessaries prepared bydirect compression of the resulting mixture.

EXAMPLE 8

Preparation of1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine

Method A: (±)-cis and (±)-trans 2-benzoyloxymethyl-5-(N₄-acetylcytosin-1-yl)-1,3-oxathiolane are prepared and separated to the(±)-cis and (±)-trans isomers as described in European Patent (EP)Specification 0 382 526. (See U.S. Pat. No. 5,047,407.) The (±)-cisisomer is fluorinated with trifluoromethyl hypofluorite influorotrichloromethane (CCl₃ F) and chloroform at -78° C., according tothe method of Robins, et al. Nucleic Acid Chemistry, Part 2, 895-900(1978). The N₄ -acetyl and 2-benzoyl groups are removed withdimethylamine in ethanol, and the product,(±)-cis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine, isisolated.

Method B: (±)-cis and (±)-trans2-benzoyloxymethyl-5-(uracil-1-yl)-1,3-oxathiolane are prepared asdescribed in EP 0 382 526). After deprotection of the 2-hydroxyl groupwith saturated methanolic ammonia, the isomers are separated on silicagel using EtOAc/MeOH as eluant (EP 0 382 526). The (±)-cis isomer isreacted with acetic anhydride in pyridine at room temperature to givethe 2-acetate. Solvent is removed in vacuo at <30° C. The 2-acetate isthen dissolved in CHCl₃ and washed with aqueous bicarbonate. Theseparated organic layer is dried, and CHCl₃ is evaporated in vacuo.(±)-cis-2-Acetyl-oxymethyl-5-(uracil-1-yl)-1,3-oxathiolane isfluorinated as described above (Method A) by the method of Robins et al.Conversion of the 5-F-uracil base to the 5-F-cytosine base is carriedout by preparation of the 4-(1,2,4-triazol-1-yl) derivative according tothe methods of C. B. Reese, J. Chem. Soc., Perkins I, 1171 (1984) and W.L. Sung, Nucleic Acids Res., 9, 6139 (1981), using 1,2,4-triazole and 2equivalents of 4-chlorophenyldichlorophosphate in dry pyridine atambient temperature. This conversion is followed by reaction withmethanol previously saturated with ammonia at 0° C., and the 2-acetateis hydrolyzed to give(±)-cis-1-(2-hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine.

Antiviral Activity

Combinations according to the invention were tested for anti-HIVactivity in an HIV-infected MT4 cell assay as described in Averett, D.R., J. Virol. Methods, 23, 263-276 (1989). The cells were exposed to HIVfor one hour prior to addition of antiviral component(s). Componentswere tested in serial 2.5-fold dilutions. After five days of incubationat 37° C., the cell number was determined. Inhibition of HIV-inducedcytopathic effect was calculated, and synergism was determined by FICplots as described by Elion, Singer, and Hitchings, J. Biol. Chem. 208,477 (1954).

The fractional inhibitor concentrations (FIC) of zidovudine andcis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine werecalculated according to the method of Elion et al., supra (Table 1).

These values can be plotted on a graph from which it can be determinedthat the combination ofcis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine andzidovudine is strongly synergistic.

                  TABLE 1                                                         ______________________________________                                        Calculation of Fractional Inhibitor                                           Concentration (FIC)                                                           70% Inhibition                                                                Zidovudine                                                                              Compound 1*  FIC       FIC                                          (1M)      (1M)         Zidovudine                                                                              Compound 1                                   ______________________________________                                        0.004     2.5          0.018     0.48                                         0.01      2.0          0.045     0.38                                         0.0256    1.6          0.12      0.31                                         0.06      1.4          0.27      0.26                                         0.22      --                                                                  --        5.2                                                                 ______________________________________                                         *Compound 1 is                                                                cis1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine.           

We claim:
 1. A method for inhibiting the replication of the humanimmunodeficiency virus which comprises administering to cells containingsaid virus a first compoundCIS-1-(2-(hydroxymethyl-1,3-oxathiolan-5-yl)-6-fluorocytosine or apharmaceutically acceptable salt thereof and a second compound3'-azido-3'-deoxythymidine, the first and second compounds being presentin an amount to provide a synergistic combination.